The mosquito- and tick-borne viruses of the Flaviviridae family represent important emerging and reemerging pathogens such as Japanese encephalitis (JE), St. Louis encephalitis (SLE), West Nile (WN), and tick-borne encephalitis (TBEV) viruses that have caused severe neuroinfections with up to 40% mortality in humans in many regions of the world. WN and SLE are endemic in North America, and WN is the major cause of viral encephalitis in the USA. During the 1999-2013 outbreaks, nearly 3 million people were infected with WN in the US, with about 17,500 reported cases of encephalitis/ meningitis that resulted in 1558 deaths, majority of which occurred in elderly. No established specific antiviral therapy or a licensed human vaccine is available to date to treat and prevent SLE and WN diseases. Also, despite the use of formalin-inactivated TBEV vaccines in endemic areas of Europe and Asia, an increasing incidence of tick-borne encephalitis during the past 2 decades emphasizes the need for an alternative vaccine that will induce a more durable immunity and protection against TBEV. The pathogenesis of neurotropic flavivirus infections involves two distinct properties of the viruses: (i) neuroinvasiveness, which relates to the capacity of the virus to replicate in the peripheral organs, induce viremia, and gain entry to the central nervous system (CNS) and (ii) neurovirulence, which is the ability of the virus to infect and replicate in cells of the CNS and cause encephalitis. In the CNS, the primary targets of neurotropic flaviviruses are neurons. Thus, successful attenuation of a neurotropic virus depends on the prevention of virus entry into the CNS and restriction of its replication in the neurons. The live attenuated TBEV, SLE, or WN vaccine candidates are being developed in the Neurotropic Flaviviruses Section of the LID using a strategy based on chimerization of a neurovirulent parental virus (TBEV, SLE, or WN) with a non-neuroinvasive dengue-4 flavivirus (DEN4) and an introduction of attenuating mutations. However, these chimeric viruses containing structural protein genes of a highly virulent parental virus exhibited a moderate or high level of neurovirulence in the developing CNS of newborn or immunodeficient mice compared to that observed for widely used live attenuated yellow fever 17D vaccine. Their further attenuation is required since a live vaccine against neurotropic virus should be fully restricted for replication in the CNS. In recent years, microRNA-targeting has become an effective strategy for selective control of tissue-tropism of both DNA and RNA viruses. In FY2013, we have demonstrated that certain miRNAs expressed in the brain can control the neurotropism and pathogeneses of neurovirulent TBEV/DEN4 virus bearing perfectly complementary miRNA target sites. The insertion of a single copy of a target for a brain-specific miRNA into the genome was sufficient to prevent the development of otherwise lethal encephalitis in adult mice infected intracerebrally with a large dose of virus. However, the efficacy of miRNA-mediated inhibition of virus replication in the immature CNS of suckling mice depends on the genetic stability of the miRNA-targeted virus that can revert to a virulent phenotype. We found that the miRNA-targeting of TBEV/DEN4 genome in the 3-noncoding region (3NCR) or in the open reading frame (ORF) strongly attenuated viral neurovirulence in the developing mouse CNS. Importantly, virus escape from miRNA-mediated suppression occurs exclusively through the deletion of inserted miRNA-targets and results in the loss of the viral genome sequence located between the two most distant miRNA targets. In FY2014, we demonstrated that simultaneous miRNA co-targeting of the genome in the ORF and 3-NCR for brain-expressed miRNAs had an additive effect and produced a more potent attenuation of the TBEV/DEN4 virus compared to separate targeting of those regions. Multiple miRNA co-targeting of these two distantly located regions completely abolished the virus neurotropism as no viral replication was detected in the developing brain of neonatal mice. Furthermore, no viral antigens were detected in neurons, and neuronal integrity in the brain of mice was well preserved. Thus, we thought that the miRNA co-targeting approach can be adapted for other viruses in order to minimize their replication in a cell- or tissue-type specific manner, but most importantly, to prevent virus escape from miRNA-mediated silencing. In FY2014, principles of miRNA-targeting to control the virus replication in the CNS were applied to chimeric SLE/DEN4 and WN/DEN4 viruses. We generated SLE/DEN4 and WN/DEN4 cDNA genomes in which the miRNA targets for mir-9 and mir-124 in the E protein gene were combined with additional miRNA-targets located in three sites in the 3NCR. These viruses subsequently demonstrated to have reduced neuroinvasiveness and neurovirulence as examined by intraperitoneal or intracerebreal infection of adult or suckling mice. Since flaviviruses are transmitted in the nature by their specific arthropod vector, the enviromental safety of live attenuated vaccines is a significant concern. The TBEV/DEN4-, SLE/DEN4- and WN/DEN4-miRNA-targeted viruses are based on the DEN4 genetic background and have a potential to be transmitted from vaccinated individuals via mosquito bite. In an effort to preclude introduction of a vaccine virus into the envroment, we explored the ability of the miRNAs expressed in Aedes mosquitoes to control DEN4 growth in salivary gland and midgut tissues. Insertion of 2, 3, or 4 targets for mosquito-specific, highly expressed miRNAs (aae-mir-184 and aae-mir-275) into the ORF and 3NCR restricts DEN4 replication in three mosquito cell lines and was enough to block the virus infectivity and dissemination in Ae. aegypti and Ae. albopictus mosquitoes, which are the competent vectors for many arboviruses, including DEN viruses. Moreover, the combined co-targeting of the DEN4 genome for mosquito-specific miRNAs and miRNAs expressed in the brain of mice resulted in the complete silencing of DEN4 replication in two different species: mosquitoes and the CNS of mice. These results suggest that chimeric virus vaccines constructed on DEN4-genetic background with mosquito- and CNS-specific miRNA targets would not be transmitted from vaccinees to other hosts via mosquitoes and would be safe for both the recipient and the environment. The upsurge of WN human infections in 2012 and 2013 suggests that the US can expect periodic WN outbreaks in the future. Availability of safe and effective vaccines against WN in endemic areas, particularly for aging populations that are at high risk for West Nile neuroinvasive disease, could be beneficial. WN/DEN4d30 is a live, attenuated chimeric vaccine against WN developed in the NFS and was found to be well-tolerated, safe, and induced a potent and durable WN antibody response in healthy adult volunteers in Phase I clinical trials. In addition, clinical and virological data, as well as results of histopathological analysis, demonstrate that the WN vaccine virus is sufficiently attenuated for the CNS of nonhuman primates as compared to an attenuated flavivirus surrogate reference (i.e., yellow fever YF 17D vaccine). Because the majority of WN neuroinvasive disease occurs in persons older than 60, we initiated a Phase I clinical trial in 2014 at the Johns Hopkins School of Hygiene and Public Health in the at-risk group of elderly volunteers. These studies were designed in order to evaluate the safety and immunogenicity of a single dose of WN vaccine in the adults >50 years of age and determine if a second dose of vaccine will improve either the seroconversion frequency or the durability of the antibody response.